Methods of detecting specific nucleic acids are of ever increasing importance in the field of molecular biology, diagnostics, and medicine. There currently exist several methods for detecting and identifying nucleic acids within biological samples. The reasons for selecting one method over another are varied, and include, among others, a desire to avoid radioactive materials, the cost or availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity for a certain application, the ease of analysis, or the ability to automate the process.
The detection of specific nucleic acids may constitute a portion of a process or an end in itself. There are many applications for the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify nucleic acids is useful in detecting microorganisms, viruses, biological molecules, and genetic expression level assays, and thus affects many fields, including human and veterinary medicine, food processing, and environmental testing.
Perhaps the most promising use of nucleic acid detection is the identification of infectious bacterial, viral, or fungal agents within biological samples of tissue, sputum, urine, blood, semen, or saliva. Timely detection of such infectious agents gives medical professionals the ability to diagnose and treat illnesses related to the infectious agents.
A number of techniques have been developed that can detect the presence of infectious agents, including viruses, bacteria, or fungi by the presence of their DNA and RNA using molecular biology approaches. Many methods detect the DNA and RNA by hybridizing their unique nucleic acids with a labeled nucleic acid detector and measuring some aspect of the detector that changes when the detector is hybridized.
Hybridization methods depend upon knowledge of the target nucleic acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which a complementary nucleic acid probe is hybridized or annealed to the nucleic acid in the sample or on a blot, and the hybridized probes are detected.
A common process for the detection of target nucleic acid takes advantage of polymerase chain reaction (“PCR”), a technique that is well known in the art. In PCR, nucleic acid primers are added to a biological sample suspected of containing a specific DNA sequence. The primers are complementary to a part of the target sequence and anneal to the denatured target if it is present within the sample. Addition of DNA polymerase extends the primer and eventually forms a DNA duplex from the initial target strand. The duplex is denatured and the process is repeated until large numbers of the target have been reproduced, which amplifies the detectability of the sequence. The amplified nucleic acid product may then be detected in a number of ways, one of which is by incorporating a labeled nucleotide into the amplified strand.
Although PCR is an effective method of assisting in the detection of a nucleic acid target having a known sequence, PCR is time consuming and adds additional complexity to the detecting method and, therefore, may not be available as a cost effective or high speed technique of testing. PCR is also subject to errors that can accumulate over progressive amplification cycles.
Alternative techniques to PCR are known that make use of dual-labeled probes known as molecular beacons. Molecular beacons maintain a closed conformation when not hybridized to a target sequence and extend in an open conformation when hybridized to a target. One end of the molecular beacon is attached to a fluorophore label, and the other end of the beacon is attached to a quencher. The beacon is initially presented in a closed conformation, where the fluorophore and quencher are adjacent to one another and tend to cancel out one another. If a target nucleic acid is present within a sample, the beacon opens and bonds to the target along the length of the target sequence. Because hybridization with a target maintains the beacon in an open conformation, the fluorophore is spaced apart from and uninhibited by the quencher, thereby fluorescing and indicating the presence of the target.
Interaction of the fluorophores and quencher molecules is often hard to detect reliably. The difference in emission between the unhybridized beacons and the hybridized beacons can sometimes be difficult to detect in the presence of background fluorescence even though molecular beacons are quite specific and able to detect sequences differing by as little as one nucleotide. Difficulty in detection makes the molecular beacons unsuitable for use in nucleic acid analysis without the use of sophisticated detection equipment.
It is desired to provide a nucleic acid detector and method of reliably detecting specific sequences of nucleic acid within a biological sample without the need to amplify the target prior to or during detection. It is desired to provide a detector and method to detect specific nucleic acid sequences that reliably indicate the presence of the target without concern of inaccuracies due to background noise. It is desired to provide a detector and method that may be used to simultaneously detect the presence of multiple targets within a sample.